Polymer conjugates ophthalmic devices comprising collagen-polymer conjugates

ABSTRACT

Pharmaceutically acceptable, non-immunogenic compositions are formed by covalently binding atelopeptide collagen to pharmaceutically pure, synthetic, hydrophilic polymers via specific types of chemical bonds to provide collagen/polymer conjugates. The atelopeptide collagen can be type I, type II or type III and may be fibrillar or non-fibrillar. The synthetic hydrophilic polymer may be polyethylene glycol and derivatives thereof having a weight average molecular weight over a range of from about 100 to about 20,000. The compositions may include other components such as liquid, pharmaceutically acceptable, carriers to form injectable formulations, and/or biologically active proteins such as growth factors. The collagen-polymer conjugates of the invention generally contain large amounts of water when formed. The conjugates can be dehydrated to form a relatively solid object. The dehydrated, solid object can be ground into particles which can be suspended in a non-aqueous fluid such as an oil and injected into a living being for the purpose of providing soft tissue augmentation. Once in place, the particles rehydrate and expand in size five fold or more.

This application is a divisional of U.S. Ser. No. 08/368,874, filed 05Jan. 1995 and now U.S. Pat. No. 5,446,051 which is a divisional of U.S.Ser. No. 08/198,128, filed 17 Feb. 1994, now issued U.S. Pat. No.5,413,791; which is a divisional of U.S. Ser. No. 07/922,541, filed 30Jul. 1992, now issued U.S. Pat. No. 5,328,955; which is acontinuation-in-part of U.S. Ser. No. 07/433,441, filed 14 Nov. 1989,now issued U.S. Pat. No. 5,162,430; which is a continuation-in-part ofU.S. Ser. No. 07/274,071, filed 21 Nov. 1988, now abandoned.

DESCRIPTION

1. Technical Field

This invention relates to collagen conjugates and specifically topharmaceutically acceptable, non-immunogenic, compositions comprisingcollagen modified by conjugation with synthetic hydrophilic polymerssuch as polyethylene glycol (PEG) wherein-the collagen and PEG arecovalently bound via specific linkages such as ether linkages.

2. Background of the Invention

Collagen is the major protein component of bone, cartilage, skin, andconnective tissue in animals. Collagen in its native form is typically arigid, rod-shaped molecule approximately 300 nm long and 1.5 nm indiameter. It is composed of three collagen polypeptides which form atight triple helix. The collagen polypeptides are characterized by along midsection having the repeating sequence -Gly-X-Y-, where X and Yare often proline or hydroxyproline, bounded at each end by the"telopeptide" regions, which constitute less than about 5% of themolecule. The telopeptide regions of the collagen chains are typicallyresponsible for the cross-linking between chains, and for theimmunogenicity of the protein Collagen occurs in several "types", havingdiffering physical properties. The most abundant types are Types I-III.

Collagen is typically isolated from natural sources, such as bovinehide, cartilage, or bones. Bones are usually dried, defatted, crushed,and demineralized to extract collagen, while hide and cartilage areusually minced and digested with proteolytic enzymes (other thancollagenose). As collagen is resistant to most proteolytic enzymes, thisprocedure conveniently serves to remove most of the contaminatingprotein found with collagen.

Collagen may be denatured by boiling, which produces the familiarproduct gelatin.

Daniels et al, U.S. Pat. No. 3,949,073, disclosed the preparation ofsoluble collagen by dissolving tissue in aqueous acid, followed byenzymatic digestion. The resulting atelopeptide collagen is soluble, andsubstantially less immunogenic than unmodified collagen. It may beinjected into suitable locations of a subject with a fibril-formationpromoter (described as a polymerization promoter in the patent) to formfibrous collagen implants in situ, for augmenting hard or soft tissue.This material is now commercially available from Collagen Corporation(Polo Alto, Calif.) under the trademark Zyderm® collagen implant.

Luck et al, U.S. Pat. No. 4,488,911, disclosed a method for preparingcollagen in solution (CIS), wherein native collagen is extracted fromanimal tissue in dilute aqueous acid, followed by digestion with anenzyme such as pepsin, trypsin, or Pronase®. The enzyme digestionremoves the telopeptide portions of the collagen molecules, providing"atelopeptide" collagen in solution. The atelopeptide CIS so produced issubstantially non-immunogenic, and is also substantiallynon-cross-linked to loss of the primary crosslinking regions. The CISmay then be precipitated by dialysis in a moderate shear environment toproduce collagen fibers which resemble native collagen fibers. Theprecipitated, reconstituted fibers may additionally be crosslinked usinga chemical agent (for example aldehydes such as formaldehyde andglutaraldehyde), or using heat or radiation. The resulting products aresuitable for use in medical implants due to their biocompatabilityoandreduced immunogenicity.

Wallace et al, U.S. Pat. No. 4,424,208, disclosed an improved collagenformulation suitable for use in soft tissue augmentation. Wallace'sformulation comprises reconstituted fibrillar atelopeptide collagen (forexample, Zyderm® collagen) in combination with particulate, crosslinkedatelopeptide collagen dispersed in an aqueous medium. The addition ofparticulate crosslinked collagen improves the implant's persistence, orability to resist shrinkage following implantation.

Smestad et al, U.S. Pat. No. 4,582,640, disclosed a glutaraldehydecrosslinked atelopeptide CIS preparation (GAX) suitable for use inmedical implants. The collagen is crosslinked under conditions favoringintrafiber bonding rather than interfiber bonding, and provides aproduct with higher persistence than non-cross-linked atelopeptidecollagen, and is commercially available from Collagen Corporation underthe trademark Zyplast® Implant.

Nguyen et al, U.S. Pat. No. 4,642,117, disclosed a method for reducingthe viscosity of atelopeptide CIS by mechanical shearing. Reconstitutedcollagen fibers are passed through a fine-mesh screen until viscosity isreduced to a practical level for injection.

Nathan et al, U.S. Pat. No. 4,563,350, disclosed osteoinductive bonerepair compositions comprising an osteoinductive factor, at least 5%nonreconstituted (afibrillar) collagen, and the remainder reconstitutedcollagen and/or mineral powder (e.g., hydroxyapatite). CIS may be usedfor the nonreconstituted collagen, and Zyderm® collagen implant (ZCI) ispreferred for the reconstituted collagen component. The material isimplanted in bone defects or fractures to speed ingrowth of osteoclastsand promote new bone growth.

Chu, U.S. Pat. No. 4,557,764, disclosed a "second nucleation" collagenprecipitate which exhibits a desirable malleability and putty-likeconsistency. Collagen is provided in solution (e.g., at 2-4 mg/mL), anda "first nucleation product" is precipitated by rapid titration andcentrifugation. The remaining supernatant (containing the bulk of theoriginal collagen) is then decanted and allowed to stand overnight. Theprecipitated second nucleation product is collected by centrifugation.

Chu, U.S. Pat. No. 4,689,399, disclosed a collagen membrane preparation,which is prepared by compressing and drying a collagen gel. Theresulting product has high tensile strength.

J. A. M. Ramshaw et al, Anal Biochem (1984) 141:361-65, and PCTapplication WO87/04078 disclosed the precipitation of bovine collagen(types I, II, and III) from aqueous PEG solutions, where there is nobinding between collagen and PEG.

Werner, U.S. Pat. No. 4,357,274, disclosed a method for improving thedurability of sclero protein (e.g., brain meninges) by soaking thedegreased tissue in H₂ O₂ or PEG for several hours prior tolyophilizing. The resulting modified whole tissue exhibits increasedpersistence.

Hiroyoshi, U.S. Pat. No. 4,678,468, disclosed the preparation ofpolysiloxane polymers having an interpenetrating network ofwater-soluble polymer dispersed within. The water-soluble polymer may bea collagen derivative, and the polymer may additionally include heparin.The polymers are shaped into artificial blood vessel grafts, and aredesigned to prevent clotting.

Other patents disclose the use of collagen preparations with bonefragments or minerals. For example, Miyata et al, U.S. Pat. No.4,314,380 disclosed a bone implant prepared by baking animal bonesegments, and soaking the baked segments in a solution of atelopeptidecollagen. Deibig et al, U.S. Pat. No. 4,192,021 disclosed an implantmaterial which comprises powdered calcium phosphate in a pastyformulation with a biodegradable polymer (which may be collagen).Commonlyowned copending U.S. patent application Ser. No. 855,004, filed22 Apr. 1986, disclosed a particularly effective bone repair materialcomprising autologous bone marrow, collagen, and particulate calciumphosphate in a solid, malleable formulation.

There are several references in the art to proteins modified by covalentconjugation to polymers, to alter the solubility, antigenicity andbiological clearance of the protein. For example, U.S. Pat. No.4,261,973 disclosed the conjugation of several allergans to PEG or PPG(polypropylene glycol) to reduce the proteins' immunogenicity. U.S. Pat.No. 4,301,144 disclosed the conjugation of hemoglobin with PEG and otherpolymers to increase the protein's oxygen carrying capability. EPO98,110 disclosed coupling an enzyme or interferon to apolyoxyethylene-polyoxypropylene (POE-POP) block polymer increases theprotein's halflife in serum. U.S. Pat. No. 4,179,337 disclosedconjugating hydrophilic enzymes and insulin to PEG or PPG to reduceimmunogenicity. Davis et al, Lancet (1981) 2:281-83 disclosed the enzymeuricase modified by conjugation with PEG to provide uric acid metabolismin serum having a long halflife and low immunogenicity. Nishida et al, JPharm Pharmacol (1984) 36:354-55 disclosed PEG-uricase conjugatesadministered orally to chickens, demonstrating decreased serum levels ofuric acid. Inada et al, Biochem & Biophys Res Comm (1984) 122:845-50disclosed lipoprotein lipase conjugation with PEG to render it solublein organic solvents. Takahashi et al, Biochem & Biophys Res Comm (1984)121:261-65 disclosed HRP conjugated with PEG to render the enzymesoluble in benzene. Abuchowski et al, Cancer Biochem Biophys (1984)7:175-86 disclosed that enzymes such as asparaginase, catalase, uricase,arginase, trypsin, superoxide dismutase, adenosine deaminase,phenylalanine ammonia-lyase, and the like, conjugated with PEG exhibitlonger half-lives in serum and decreased immunogenicity. However, thesereferences are essentially concerned with modifying the solubility andbiological characteristics of proteins administered in lowconcentrations in aqueous solution.

M. Chvapil et al, J Biomed Mater Res (1969) 3:315-32 disclosed acomposition prepared from collagen sponge and a crosslinked ethyleneglycol monomethacrylate-ethylene glycol dimethacrylate hydrogel. Thecollagen sponge was prepared by lyophilizing an aqueous mixture ofbovine hide collagen and methylglyoxal (a tanning agent). Thesponge-hydrogel composition was prepared by polymerizing ethylene glycolmonomethacrylate and ethylene glycol dimethacrylate in the sponge.

SUMMARY OF THE INVENTION

Pharmaceutically acceptable, non-immunogenic compositions are formed bycovalently binding atelopeptide collagen to pharmaceutically pure,synthetic, hydrophilic polymers via specific types of chemical bonds toprovide collagen/polymer conjugates. The atelopeptide collagen can betype I, type II or type III and may be fibrillar or non-fibrillar. Thesynthetic hydrophilic polymer may be polyethylene glycol and derivativesthereof having a weight average molecular weight over a range of fromabout 100 to about 20,000. The compositions may include other componentssuch as pharmaceutically acceptable fluid carriers to form injectableformulations, and/or biologically active proteins such as cytokines. Thecollagen-polymer conjugates of the invention generally contain largeamounts of water when formed. The conjugates can be dehydrated to form arelatively solid object. The dehydrated, solid object can be ground intoparticles which can be suspended in a non-aqueous fluid and injectedinto a living being for the purpose of soft tissue augmentation. Once inplace, the particles rehydrate and expand in size five fold or more.

The essence of the invention relates to the collagen/polymer conjugateswhich are applied and used in a variety of medical and pharmaceuticalapplications. Although all aspects of the invention generally relate tothese conjugates, the invention can be categorized into the followingeight different embodiments: (1) the most basic embodiment includes thecollagen/polymer conjugates and pharmaceutical compositions which areformulated using these conjugates, which compositions includepharmaceutically acceptable carriers in different types and amounts. (2)One of the most important uses for the conjugates and compositions ofthe invention is in. methods of effecting soft tissue augmentation. Thecompositions are formulated in a flowable form and injected intopatients, such as into facial areas, in order to provide for soft tissueaugmentation. The method can be varied so that the reaction between thecollagen and the polymer occurs in situ. Furthermore, the conjugates canbe dehydrated and then ground into particles, suspended in an inert,non-aqueous carrier, and injected into a patient. After injection, thecarrier will be removed by natural physiological conditions and theparticles will rehydrate and swell to their original size. (3) All typesof conjugates and conjugate compositions of the invention can becombined with various types of cytokines. The cytokines may be eithersimply admixed with the PEG-collagen conjugate or chemically conjugatedto di- or multifunctional PEG-collagen (collagen-PEG-cytokine). In thecase of an admixture, the cytokines are not chemically bound to thePEG-collagen and may migrate away from the site of administration intothe surrounding tissue providing for sustained release and localtherapeutic effects. In the case of the cytokine that is chemicallyconjugated to the collagen-polymer, the cytokine retains its biologicalactivity even while bound to the conjugate. (4) The various conjugatesand compositions of the invention can be further combined with particlesand materials in order to increase the structural integrity of thecompositions so that they can be used in the augmentation of hard tissuesuch as bone and cartilage. (5) The conjugates and compositionscontaining the conjugates can be coated on to various medical devices,including catheters, bone implants, and platinum wires to treataneurysms. (6) The conjugates can be formulated into various ophthalmicdevices, such as lenticules or corneal shields. (7) The reactionsbetween the collagen and the polymer can be designed in a manner so asto form tubular, cylindrical, or spherical shapes for use as nervegrowth tubes, blood vessel grafts or breast implant shells. (8) Theconjugates and conjugate formulations of the invention can be covalentlybound to the chemically derivatized surface of silicon breast implantsto prevent capsular contracture and formation of scar tissue.

A primary object of the invention is to provide collagen-polymerconjugates formed by covalently binding polymers such as polyethyleneglycol to collagen.

Another object of the invention is to provide pharmaceuticallyacceptable, non-immunogenic compositions comprising pharmaceuticallyacceptable liquid carriers having collagen-polymer conjugates therein.

Another object of the invention is to provide a method of tissueaugmentation comprising forming the collagen-polymer conjugates,dehydrating the conjugates to form a solid, grinding the solid intoparticles, suspending the particles in a liquid non-aqueous carrier andinjecting the suspension into the site of augmentation after which theparticles will rehydrate and expand in size.

An important advantage of the present invention is that thecollagen-polymer conjugates have a high degree of stability over longperiods of time under physiological conditions.

A feature of the invention is that the conjugates can be formed using arange of different molecular weight polymers in order to adjust physicalcharacteristics of the resulting composition.

Another advantage of the present invention is that the collagen-polymerconjugates have superior handling characteristics as compared withconventional collagen compositions.

Another advantage of the present invention is that the collagen-polymerconjugate compositions generate a decreased immune reaction as comparedwith conventional pharmaceutically acceptable collagen compositions andcollagen compositions crosslinked by other means, such as heat,irradiation, or glutaraldehyde treatment.

Other advantages and features of the present invention is that thecollagen-polymer compositions have improved moldability, malleability,and elasticity as compared with conventional collagen compositions.

Other features of the present invention include the ability to formulatethe compositions and conjugates in combination with pharmaceuticallyactive molecules such as cytokines in order to improve the activity andavailable half-life of such cytokines under physiological conditions.

Another feature of the present invention is that the collagen is boundto the polymer by means of a convalent ether linkage.

Another advantage of the present invention is that due to the presenceof the ether linkage, the covalent bond between the collagen and thepolymer is resistant to breakage due to hydrolysis.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis, and usage of thecollagen-polymer conjugates as more fully set forth below, referencebeing made to the attached figures and included specific examples andformulations forming a part hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the force necessary to extrude three compositions:Zyderm® collagen implant (ZCI), a glutaraldehyde-crosslinked collagen(GAX), and a collagen-PEG conjugate of the invention.

FIG. 2 illustrates the results of the experiment conducted in Example6E, demonstrating the retention of biologically active TGF-β1 in acrosslinked collagen-dPEG composition.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Before the present collagen-polymer conjugates and processes for makingand using such are described, it is to be understood that this inventionis not limited to the particular conjugates or processes and methodsdescribed as such may, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting as the scope of thepresent invention will be limited only by the appended claims.

It must be noted that, as used in this specification and the appendedclaims, the singular forms "a", "an" and "the" include plural referrentsunless the context clearly dictates otherwise. Thus, for example,reference to "a polymer" includes-mixtures of polymers reference to "anamino group" includes one or more different types of amino groups knownto those skilled in the art and reference to "the collagen" includesmixtures of different types of collagens and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein may be usefulin the practice or testing of the present invention, preferred methodsand materials are described below. All publications mentioned herein areincorporated herein by reference. Further, specific terminology ofparticular importance to the description of the present invention isdefined below.

A. Definitions

The term "collagen" as used herein refers to all forms of collagen,including those which have been processed or otherwise modified.Preferred collagens are treated to remove the immunogenic telopeptideregions ("atelopeptide collagen"), are soluble, and may be in thefibrillar or non-fibrillar form. Type I collagen is best suited to mostapplications involving bone or cartilage repair. However, other forms ofcollagen are also useful in the practice of the invention, and are notexcluded from consideration here. Collagen crosslinked using heat,radiation, or chemical agents such as glutaraldehyde may be conjugatedwith polymers as described herein to form particularly rigidcompositions. Collagen crosslinked using glutaraldehyde or other(nonpolymer) linking agents is referred to herein as "GAX", whilecollagen crosslinked using heat and/or radiation is termed "HRX."Collagen used in connection with the preferred embodiments of theinvention is in a pharmaceutically pure form such that it can beincorporated into a human body for the intended purpose.

The term "synthetic hydrophilic polymer" as used herein refers to asynthetic polymer having an average molecular weight and compositionwhich renders the polymer essentially water-soluble. Preferred polymersare highly pure or are purified to a highly pure state such that thepolymer is or is treated to become pharmaceutically pure. Mosthydrophilic polymers can be rendered water-soluble by incorporating asufficient number of oxygen (or less frequently nitrogen) atomsavailable for forming hydrogen bonds in aqueous solutions. Preferredpolymers are hydrophilic but not soluble. Hydrophilic polymers usedherein include polyethylene glycol, polyoxyethylene, polymethyleneglycol, polytrimethylene glycols, polyvinylpyrrolidones, and derivativesthereof. The polymers can be linear or multiply branched and will not besubstantially cross-linked. Other suitable polymers includepolyoxyethylene-polyoxypropylene block polymers and copolymers.Polyoxyethylene-polyoxypropylene block polymers having an ethylenediamine nucleus (and thus having four ends) are also available and maybe used in the practice of the invention. Naturally occurring polymerssuch as proteins, starch, cellulose, heparin, and the like are expresslyexcluded from the scope of this definition although the inventionincludes polymer mixtures with naturally occurring polymers therein. Allsuitable polymers will be non-toxic, non-inflammatory andnon-immunogenic when administered subcutaneously, and will preferably beessentially nondegradable in vivo over a period of at least severalmonths. The hydrophilic polymer may increase the hydrophilicity of thecollagen, but does not render it water-soluble. Presently preferredhydrophilic polymers are mono-, di-, and multifunctional polyethyleneglycols (PEG). Monofunctional PEG has only one reactive hydroxy group,while difunctional PEG has reactive groups at each end. MonofunctionalPEG preferably has a weight average molecular weight between about 100and about 15,000, more preferably between about 200 and about 8,000, andmost preferably about 4,000. Difunctional PEG preferably has a molecularweight of about 400 to about 40,000, more preferably about 3,000 toabout 10,000. Multifunctional PEG preferably has a molecular weightbetween about 3,000 and 100,000.

PEG can be rendered monofunctional by forming an alkylene ether at oneend. The alkylene ether may be any suitable alkoxy radical having 1-6carbon atoms, for example, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,hexyloxy, and the like. Methoxy is presently preferred. Difunctional PEGis provided by allowing a reactive hydroxy group at each end of thelinear molecule. The reactive groups are preferably at the ends of thepolymer, but may be provided along the length thereof. Polyfunctionalmolecules are capable of crosslinking the compositions of the invention,and may be used to attach cytokines to collagen.

The term "chemically conjugated" as used herein means attached through acovalent chemical bond. In the practice of the invention, a synthetichydrophilic polymer and collagen may be chemically conjugated by using alinking radical, so that the polymer and collagen are each bound to theradical, but not directly to each other. The term "collagen-polymer"refers to collagen chemically conjugated to a synthetic hydrophilicpolymer, within the meaning of this invention. Thus, "collagen-PEG" (or"PEG-collagen") denotes a composition of the invention wherein collagenis chemically conjugated to PEG. "Collagen-dPEG" refers to collagenchemically conjugated to difunctional PEG, wherein the collagenmolecules are typically crosslinked. "Crosslinked collagen" refers tocollagen in which collagen molecules are linked by covalent bonds withpolyfunctional (including difunctional) polymers. Terms such as"GAX-dPEG" and "HRX-dPEG" indicate collagen crosslinked by both adifunctional hydrophilic polymer and a crosslinking agent such asglutaraldehyde or heat. The polymer may be "chemically conjugated" tothe collagen by means of a number of different types of chemicallinkages. For example, the conjugation can be via an ester or urethanelinkage, but is more preferably by means of an ether linkage. An etherlinkage is preferred in that it can be formed without the use of toxicchemicals and is not readily susceptible to hydrolysis in vivo.

Those of ordinary skill in the art will appreciate that syntheticpolymers such as polyethylene glycol Cannot be prepared practically tohave exact molecular weights, and that the term "molecular weight" asused herein refers to the weight average molecular weight of a number ofmolecules in any given sample, as commonly used in the art. Thus, asample of PEG 2,000 might contain a statistical mixture of polymermolecules ranging in weight from, for example, 1,500 to 2,500 daltonswith one molecule differing slightly from the next over a range.Specification of a range of molecular weight indicates that the averagemolecular weight may be any value between the limits specified, and mayinclude molecules outside those limits. Thus, a molecular weight rangeof about 800 to about 20,000 indicates an average molecular weight of atleast about 800, ranging up to about 20 kDa.

The term "available lysine residue" as used herein refers to lysine sidechains exposed on the outer surface of collagen molecules, which arepositioned in a manner to allow reaction with activated PEG. The numberof available lysine residues may be determined by reaction with sodium2,4,6-trinitrobenzenesulfonate (TNBS).

The terms "treat" and "treatment" as used herein refer to augmentation,repair, prevention, or alleviation of defects, particularly defects dueto loss or absence of soft tissue or soft tissue support, or to loss orabsence of bone. Additionally, "treat" and "treatment" also refer to theprevention, maintenance, or alleviation of disorders or disease using abiologically active protein coupled to the collagen-polymer compositionof the invention. Accordingly, treatment of soft tissue includesaugmentation of soft tissue, for example implantation ofcollagen-polymer conjugates of the invention to restore normal ordesirable dermal contours, as in the removal of dermal creases orfurrows, or as in the replacement of subcutaneous fat in maxillary areaswhere the fat is lost due to aging, or in the augmentation of submucosaltissue such as the urinary or lower esophageal sphincters. Treatment ofbone and cartilage includes the use of collagen-polymer conjugates, andparticularly collagen-PEG in combination with suitable particulatematerials, to replace or repair bone tissue, for example, in thetreatment of bone non-unions or fractures. Treatment of bone alsoincludes use of cartilaginoid collagen-dPEG compositions, with orwithout additional bone growth factors. Compositions comprisingcollagen-polymer with ceramic particles, preferably hydroxyapatiteand/or tricalcium phosphate, are particularly useful for the repair ofstress-bearing bone due to its high tensile strength.

The term "cytokine" is used to describe biologically active moleculesincluding growth factors and active peptides which aid in healing orregrowth of normal tissue. The function of cytokines is two-fold: 1)they can incite local cells to produce new collagen or tissue, or 2)they can attract cells to the site in need of correction. As such,cytokines serve to encourage "biological anchoring" of the collagenimplant within the host tissue. As previously described, the cytokinescan either be admixed with the collagen-polymer conjugate or chemicallycoupled to the conjugate. For example, one may incorporate cytokinessuch as epidermal growth factor (EGF), transforming growth factor (TGF)alpha, TGF-β (including any combination of TGF-βs), TGF-β1, TGF-β2,platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidicfibroblast growth factor (FGF), basic FGF, connective tissue activatingpeptides (CTAP), β-thrombo-globulin, insulin-like growth factors, tumornecrosis factor (TNF), interleukins, colony stimulating factors (CSFs),erythropoietin (EPO), nerve growth factor (NGF), interferons (IFN) bonemorphogenic protein (BMP), osteogenic factors, and the like.Incorporation of cytokines, and appropriate combinations of cytokinescan facilitate the regrowth and remodeling of the implant into normalbone tissue, or may be used in the treatment of wounds. Furthermore, onemay chemically link the cytokines to the collagen-polymer composition byemploying a suitable amount of multifunctional polymer molecules duringsynthesis. The cytokines may then be attached to the free polymer endsbythe same method used to attach PEG to collagen, or by any other suitablemethod. By tethering cytokines to the implant, the effective amount ofcytokine is substantially reduced. Dried collagen-PEG compositionshaving sponge-like characteristics may be prepared as wound dressings,or when incorporated with cytokines, they serve as effectivecontrolled-release drug delivery matrices. By varying the chemicallinkage between the collagen and the synthetic polymer, it is possibleto vary the effect with respect to the release of the cytokine. Forexample, when a "ester" linkage is used, the linkage is more easilybroken under physiological conditions, allowing for sustained release ofthe growth factor from the matrix. However, when an "ether" linkage isused, the bonds are not easily broken and the cytokine will remain inplace for longer periods of time with its active sites exposed providinga biological effect on the natural substrate for the active site of theprotein. It is possible to include a mixture of conjugates withdifferent linkages so as to obtain variations in the effect with respectto the release of the cytokine, i.e., the sustained release effect canbe modified to obtain the desired rate of release.

The term "effective amount" refers to the amount of composition requiredin order to obtain the effect desired. Thus, a "tissue growth-promotingamount" of a composition containing a cytokine refers to the amount ofcytokine needed in order to stimulate tissue growth to a detectabledegree. Tissue, in this context, includes connective tissue, bone,cartilage, epidermis and dermis, blood, and other tissues. The actualamount which is determined to be an effective amount will vary dependingon factors such as the size, condition, sex and age of the patient andcan be more readily determined by the caregiver.

The term "sufficient amount" as used herein is applied to the amount ofcarrier used in combination with the collagen-polymer conjugates of theinvention. A sufficient amount is that amount which, when mixed with theconjugate, renders it in the physical form desired, for example,injectable solution, injectable suspension, plastic or malleableimplant, rigid stress-bearing implant, and so forth. Injectableformulations generally include an amount of a carrier sufficient torender the composition smoothly injectable without significant need tointerrupt the injection process, whereas malleable implants containsubstantially less carrier and have a clay-like consistency. Rigidstress-bearing implants may include no carrier at all and have a highdegree of structural integrity. The amount of the carrier can be variedand adjusted depending on the particular conjugate used and the endresult desired. Such adjustments will be apparent to those skilled inthe art.

The term "suitable particulate material" as used herein refers to aparticulate material which is substantially insoluble in water,non-immunogenic, biocompatible, and immiscible with collagen-polymerconjugate. The particles of material may be fibrillar, or may range insize from about 20 to 250 μm in diameter and be bead-like or irregularin shape. Exemplary particulate materials include without limitationfibrillar crosslinked collagen, gelatin beads, crosslinked collagen-dPEGparticles, polytetrafluoroethylene beads, silicone rubber beads,hydrogel beads, silicon carbide beads, and glass beads. Preferredparticulate materials are calcium phosphates, most preferably,hydroxyapatite and/or tricalcium phosphate.

The term "solid implant" refers to any solid object which is designedfor insertion and use within the body, and includes bone and cartilageimplants (e.g., artificial joints, retaining pins, cranial plates, andthe like, of metal, plastic and/or other materials), breast implants(e.g., silicone gel envelopes, foam forms, and the like), catheters andcannulas intended for long-term use (beyond about three days) in place,artificial organs and vessels (e.g., artificial hearts, pancreases,kidneys, blood vessels, and the like), drug delivery devices (includingmonolithic implants, pumps and controlled release devices such as Alzet®minipumps, steroid pellets for anabolic growth or contraception, and thelike), sutures for dermal or internal use, periodontal membranes,ophthalmic shields, corneal lenticules, and the like.

The term "suitable fibrous material", as used herein, refers to afibrous material which is substantially insoluble in water,non-immunogenic, biocompatible, and immiscible with the collagen/polymerconjugate of the invention. The fibrous material may comprise a varietyof materials having these characteristics and are combined withcompositions of the collagen/polymer conjugate in order to form and/orprovide structural integrity to various implants or devices used inconnection with medical and pharmaceutical uses. For example, thecollagen/polymer conjugate compositions of the invention can be coatedon the "suitable fibrous material", which can then be wrapped around abone to provide structural integrity to the bone. Thus, the "suitablefibrous material" is useful in forming the "solid implants" of theinvention.

The term "in situ" as used herein means at the site of administration.Thus, the injectable reaction mixture compositions are injected orotherwise applied to a site in need of augmentation, and allowed tocrosslink at the site of injection. Suitable sites will generally beintradermal or subcutaneous regions for augmenting dermal support, atthe site of bone fractures for wound healing and bone repair, and withinsphincter tissue for sphincter augmentation (e.g., for restoration ofcontinence).

The term "aqueous mixture" of collagen includes liquid solutions,suspensions, dispersions, colloids, and the like containing collagen andwater.

The term "NFC cartilage" as used herein refers to a composition of theinvention which resembles cartilage in physical consistency. NFCcartilage is prepared from nonfibrillar collagen (e.g., collagen insolution) and is crosslinked with a hydrophilic polymer, especiallyusing dPEG. As an artifact of the production process or by design, NFCcartilage may contain about 0-20% fibrillar collagen. NFC cartilage isgenerally prepared by adding dPEG in acidic solution to an acidicsolution of collagen, and allowing conjugation to occur prior toneutralization. The term "NFC-FC cartilage" refers to a compositionsimilar to NFC cartilage, wherein the percentage of fibrillar collagenis about 20-80%. NFC-FC.cartilage is generally prepared by adding dPEGin a neutralizing buffer to an acidic solution of collagen. Theneutralizing buffer causes collagen fibril formation during theconjugation process. Similarly, "FC cartilage" refers to a compositionof the invention which is prepared from fibrillar collagen and adifunctional hydrophilic polymer. FC cartilage may generally be preparedusing dPEG and fibrillar collagen in neutral solutions/suspensions.

B. General Method

B.1 Preparation:

To form the conjugates of the invention collagen must be chemicallybound to a synthetic hydrophilic polymer. This can be carried out in avariety of ways. In accordance wiith the preferred method, the synthetichydrophilic polymer is activated and then reacted with the collagen.Alternatively, the hydroxyl or amino groups present on the collagen canbe activated and the activated groups will react with the polymer toform the conjugate. In accordance with a less preferred method, alinking group with activated hydroxyl or amino groups thereon can becombined with the polymer and collagen in a manner so as to concurrentlyreact with both the polymer and collagen forming the conjugate. Othermethods of forming the conjugates will become apparent to those skilledin the art upon reading this disclosure. Since the conjugates of theinvention are to be used in the human body it is important that all ofthe components, including the polymer, collagen, and linking group, ifused form a conjugate that is unlikely to be rejected by the body.Accordingly, toxic and/or immunoreactive components are not preferred asstarting materials. Some preferred starting materials and methods offorming conjugates are described further below.

Although different hydrophilic synthetic polymers can be used inconnection with forming the conjugate must be biocompatible, relativelyinsoluble, but hydrophilic and is preferrably one or more forms ofpolyethylene glycol (PEG), due to its known bio-compatiblility. Variousforms of PEG are extensively used in the modification of biologicallyactive molecules because PEG can be formulated to have a wide range ofsolubilities and because it lacks toxicity, antigenicity,immunogenicity, and does not typically interfere with the enzymaticactivities and/or conformations of peptides. Further, PEG is generallynon-biodegradable and is easily excreted from most living organismsincluding humans.

The first step in forming the collagen-polymer conjugates of theinvention generally involves the functionalization of the PEG molecule.Various functionalized polyethylene glycols have been used effectivelyin fields such as protein modification (see Abuchowski et al., Enzymesas Drugs, John Wiley & Sons: New York, N.Y. (1981) pp. 367-383; andDreborg et al., Crit. Rev. Therap. Drug Carrier Syst. (1990) 6:315, bothof which are incorporated herein by reference), peptide chemistry (seeMutter et al., The Peptides, Academic: New York, N.Y. 2:285-332; andZalipsky et al., Int. J. Peptide Protein Res. (1987) 30:740, both ofwhich are incorporated herein by reference), and the synthesis ofpolymeric drugs (see Zalipsky et al., Eur. Polym. J. (1983) 19:1177; andOuchi et al., J. Macromol. Sci. Chem. (1987) A24:1011, both of which areincorporated herein by reference). Various types of conjugates formed bythe binding of polyethylene glycol with specific pharmaceutically activeproteins have been disclosed and found to have useful medicalapplications in part due to the stability of such conjugates withrespect to proteolytic digestion, reduced immunogenicity and longerhalf-lives within living organisms.

One form of polyethylene glycol which has been found to be particularlyuseful is monomethoxy-polyethylene glycol (mPEG), which can be activatedby the addition of a compound such as cyanuric chloride, then coupled toa protein (see Abuchowski et al., J. Biol. Chem. (1977) 252:3578, whichis incorporated herein by reference). Although such methods ofactivating polyethylene glycol can be used in connection with thepresent invention, they are not particularly desirable in that thecyanuric chloride is relatively toxic and must be completely removedfrom any resulting product in order to provide a pharmaceuticallyacceptable composition.

Activated forms of PEG can be made from reactants which can be purchasedcommercially. One form of activated PEG which has been found to beparticularly useful in connection with the present invention ismPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (see Abuchowski etal., Cancer Biochem. Biphys. (1984) 7:175, which is incorporated hereinby reference). Activated forms of PEG such as SS-PEG react with theproteins under relatively mild conditions and produce conjugates withoutdestroying the specific biological activity and specificity of theprotein attached to the PEG. However, when such activated PEGs arereacted with proteins, they react and form linkages by means of esterbonds. Although ester linkages can be used in connection with thepresent invention, they are not particularly preferred in that theyundergo hydrolysis when subjected to physiological conditions overextended periods of time (see Dreborg et al., Crit. Rev. Therap. DrugCarrier Syst. (1990) 6:315; and Ulbrich et al., J, Makromol. Chem.(1986) 187:1131, both of which are incorporated herein by reference).

It is possible to link PEG to proteins via urethane linkages, therebyproviding a more stable attachment which is more resistant to hydrolyticdigestion than the ester linkages (see Zalipsky et al., Polymeric Drugand Drug Delivery Systems, Chapter 10, "Succinimidyl Carbonates ofPolyethylene Glycol" (1991) incorporated herein by reference to disclosethe chemistry involved in linking various forms of PEG to specificbiologically active proteins). The stability of urethane linkages hasbeen demonstrated under physiological conditions (see Veronese et al.,Appl. Biochem. Biotechnol. (1985) 11:141; and Larwood et al., J.Labelled Compounds Radiopharm. (1984) 21:603, both of which areincorporated herein by reference). Another means of attaching the PEG toa protein can be by means of a carbamate linkage (see Beauchamp et al.,Anal. Biochem. (1983) 131:25; and Berger et al., Blood (1988) 71:1641,both of which are incorporated herein by reference). The carbamatelinkage is created by the use of carbonyldiimidazole-activated PEG.Although such linkages have advantages, the reactions are relativelyslow and may take 2 to 3 days to complete.

The various means of activating PEG described above and publications(all of which are incorporated herein by reference) cited in connectionwith the activation means are described in connection with linking thePEG to specific biologically active proteins and not collagen. However,the present invention now discloses that such activated PEG compoundscan be used in connection with the formation of collagen-PEG conjugates.Such conjugates provide a range of improved unexpected characteristicsand as such can be used to form the various compositions of the presentinvention. [Polymeric Drug and Drug Delivery Systems, Chapter 10,"Succinimidyl Carbonates of Polyethylene Glycol" (1991), incorporatedherein by reference to disclose the chemistry involved in linkingvarious forms of PEG to specific biologically active proteins.]

B.2 Specific Forms of Activated PEG.

As indicated above, the conjugates of the present invention can beprepared by covalently binding a variety of different types of synthetichydrophilic polymers to collagen. However, because the final product orconjugate obtained must have a number of required characteristics suchas being biocompatible and non-immunogenic, it has been found useful touse polyethylene glycol as the synthetic hydrophilic polymer. Thepolyethylene glycol must be modified in order to provide activatedgroups on one or preferably both ends of the molecule so that covalentbinding can occur between the PEG and the collagen. Some specificfunctionalized forms of PEG are shown structurally below, as are theproducts obtained by reacting these functionalized forms of PEG withcollagen.

The first functionalized PEG is difunctionalized PEG succinimidylglutarate, referred to herein as (SG-PEG). The structural formula ofthis molecule and the reaction product obtained by reacting it withcollagen is shown below: ##STR1##

Another difunctionally activated form of PEG is referred to as PEGsuccinimidyl (S-PEG). The structural formula for this compound and thereaction product obtained by reacting it with collagen is shown below.It should be noted that the methyl group is repeated twice. In a generalstructural formula for this compound, the is replace with an "n" and inthis embodiment, n=3, in that there are three repeating CH₈ 2 groups oneach side of the PEG. The above structure results in a conjugate whichincludes a "ether" linkage which is not subject to hydrolysis. This isdistinct from the first conjugate shown above, wherein an ester linkageis provided. The ester linkage is subject to hydrolysis underphysiological conditions. ##STR2##

Yet another derivatized form of polyethylene glycol, wherein n=2 isshown below, as is the conjugate formed by reacting the derivatized PEGwith collagen. ##STR3##

In another preferred embodiment of the invention similar to theimmediate two above formulas, is provided when n=1. The structuralformula and resulting conjugate are shown below. It is noted that theconjugate includes both an ether and a peptide linkage. These linkagesare stable under physiological conditions. ##STR4##

Yet another derivatized form of PEG is provided when n=0. Thedifunctionalized form is referred to as PEG succinimidyl carbonate(SC-PEG). The structural formula of this compound and the conjugateformed by reacting SC-PEG with collagen is shown below. Although thisconjugate includes a urethane linkage, the conjugate has been found notto have a high degree of stability under physiological conditions.##STR5##

All of the above derivatives involve the inclusion of the succinimidylgroup. However, different activating groups can be attached to one orboth ends of the PEG. For example, the PEG can be derivatized to formdifunctional PEG propion aldehyde (A-PEG), which is shown below, as isthe conjugate formed by the reaction of A-PEG with collagen. ##STR6##

Yet another functionalized form of polyethylene glycol is difunctionalPEG glycidyl ether (E-PEG), which is shown below, as is the conjugateformed by reacting such with collagen. ##STR7##

The conjugates formed using the functionalized forms of PEG varydepending on the functionalized form of PEG which is used in thereaction. Furthermore, the final product can be varied with respect toits characteristics by changing the molecular weight of the PEG. Ingeneral, the stability of the conjugate is improved by eliminating anyester linkages between the PEG and the collagen and including etherand/or urethane linkages. In certain situations, it is desirable toinclude the weaker ester linkages so that the linkages are graduallybroken by hydrolysis under physiological conditions, breaking apart thematrix and releasing a component held therein, such as a growth factor.By varying the chemical structure of the linkage, the rate of sustainedrelease can be varied.

The crosslinking reaction between the collagem and polymer may beperformed in vitro, or a reaction mixture may be injected forcrosslinking in situ. At sufficient density, crosslinkedcollagen-polymer conjugates resemble cartilage, and are useful assubstitutes thereof, (e.g., cranial onlay, ear and nose reconstruction,and the like). Polyfunctional polymers may also be used to crosslinkcollagen molecules to other proteins (e.g., glycosaminoglycans,chondroitin sulfates, fibronectin, and the like), particularly growthfactors, for compositions particularly suited for use in wound healing,osteogenesis, and immune modulation. Such tethering of cytokines tocollagen molecules provides an effective slow-release drug deliverysystem.

Suitable collagens include all types of collagen, preferably types I, IIand III. Collagens may be soluble (for example, commercially availableVitrogene 100 collagen-in-solution), and may or may not have thetelopeptide regions. Preferably, the collagen will be reconstitutedfibrillar atelopeptide collagen, for example Zyderm® collagen implant(ZCI) or atelopeptide. collagen in solution (CIS). various forms ofcollagen are available commercially, or may be prepared by the processesdescribed in, for example, U.S. Pat. Nos. 3,949,073; 4,488,911;4,424,208; 4,582,640; 4,642,117; 4,557,764; and 4,689,399, allincorporated herein by reference. Non-fibrillar, atelopeptide,reconstituted collagen is preferred in order to form certain products.

The compositions of the invention comprise collagen chemicallyconjugated to a selected synthetic hydrophilic polymer or polymers.Collagen contains a number of available amino and hydroxy groups Whichmay be used to bind the synthetic hydrophilic polymer. The polymer maybe bound using a "linking group", as the native hydroxy or amino groupsin collagen and, in the polymer frequently require activation beforethey can be linked. For example, one may employ compounds such asdicarboxylic anhydrides (e.g., glutaric or succinic anhydride) to form apolymer derivative (e.g., succinate), which may then be activated byesterification with a convenient leaving group, for example,N-hydroxysuccinimide, N,N'-disuccinimidyl oxalate, N,N'-disuccinimidylcarbonate, and the like. See also Davis, U.S. Pat. No. 4,179,337 foradditional linking groups. Presently preferred dicarboxylic anhydridesthat are used to form polymer-glutarate compositions include glutaricanhydride, adipic anhydride, 1,8-naphthalene dicarboxylic arthydride,and 1,4,5,8-naphthalenetetracarboxylic dianhydride. The polymer thusactivated is then allowed to react with the collagen, forming acollagen-polymer composition of the invention.

Conjugates with Ester Linkages

In one embodiment, a pharmaceutically pure form ofmonomethylpolyethylene glycol (mPEG) (mw 5,000) is reacted with glutaricanhydride (pure form) to create mPEG glutarate. The glutarate derivativeis then reacted with N-hydroxysuccinimide to form a succinimidylmonomethylpolyethylene glycol glutarate. The succinimidyl ester (mPEG*,denoting the activated PEG intermediate) is then capable of reactingwith free amino groups present on collagen (lysine residues) to form acollagen-PEG conjugate of the invention wherein one end of the PEGmolecule is free or nonbound. Other polymers may be substituted for themonomethyl PEG, as described above. Similarly, the coupling reaction maybe carried out using any known method for derivatizing proteins andsynthetic polymers. The number of available lysines conjugated may varyfrom a single residue to 100% of the lysines, preferably 10-50%, andmore preferably 20-30%. The number of reactive lysine residues may bedetermined by standard methods, for example by reaction with TNBS.

The resulting product is a smooth, pliable, rubbery mass having a shinyappearance. It may be wetted, but is not water-soluble. It may beformulated as a suspension at any convenient concentration, preferablyabout 30-65 mg/mL, and may be implanted by injection through a suitablesyringe. The consistency of the formulation may be adjusted by varyingthe amount of liquid used.

Compositions for Bone Repair

Formulations suitable for repair of bone defects or nonunions may beprepared by providing high Concentration compositions ofcollagen-polymer, or by admixture with suitable particulate materials.When making bone repair compositions, the linkage between the collagenand polymer must be an ether linkage in order to avoid deterioration dueto the hydrolysis of the ester linkages. Such collagen-polymerparticulate compositions may be malleable or rigid, depending on theamount of liquid incorporated. Formulations for treatment ofstress-bearing bone are preferably dried and rigid, and will generallycomprise between about 45% and 85% particuiate mineral, for examplehydroxyapatite, tricalcium phosphate, or mixtures thereof. The tensilestrength and rigidity may be further increased by heating thecomposition under vacuum at about 60°-90° C., preferably about 75° C.,for about 5 to 15 hours, preferably about 10 hours. Malleablecompositions may be used for repair of non-stressed bone or cartilage.

The activated mPEG* may be replaced, in whole or in part, bydifunctional activated PEG (dPEG,, e.g., non-methylated PEG which isthen activated at each end) thus providing a crosslinked or partiallycrosslinked collagen composition. Such compositions are, however, quitedistinct from conventionally-crosslinked collagen compositions (e.g.,using heat, radiation, glutaraldehyde, and the like), as the long-chainsynthetic hydrophilic polymer imparts a substantial hydrophiliccharacter to the composition. In a presently preferred embodiment,approximately 1-20% of the mPEG is difunctional PEG. The character ofthe composition may be adjusted as desired, by varying the amount ofdifunctional PEG included during the process.

In another presently preferred embodiment, difunctional PEG*(substantially 100% at pH 7) is used to crosslink collagen. In oneversion, CIS (about 3-100 mg/mL, preferably about 10-40 mg/mL) isallowed to react with dPEG, (difunctional PEG activated at each end byaddition of an acid anhydride having a leaving group such assuccinimide) having a molecular weight of about 2,000 to about 20,000(preferably about 3,400-10,000) which is added as a concentratedsolution to a final reaction mixture concentration of about 5-40%,preferably about 10-20%. This represents a 5- to 10-fold excess of dPEG,to collagen on a molar basis. The collagen molecules bind to dPEG*,without mechanical mixing or agitation, and settle out of solution toproduce a cartilaginoid collagen-polymer conjugate containingapproximately 20-80% fibrillar collagen. The conjugate is then washedwith PBS to remove any remaining unreacted dPEG*, providing the materialof the invention. A cartilaginoid collagen-polymer conjugate may also beprepared by mixing dPEG, solution (pH 3) with collagen-in-solutionbetween two syringes to homogeneity, and then casting into a suitablecontainer (e.g., a Petri dish). A 20% w/v dPEG* solution (pH 7) is thenadded to the non-fibrillar collagen-PEG solution to result in a lightlycartilaginoid fibrillar collagen-polymer conjugate. The resulting NFC-Fconjugate cartilage contains approximately 1-40% fibrillar collagen. Thecharacteristics of the final product may be adjusted by varying theinitial reaction conditions. In general, increased collagen and/orpolymer concentrations provide a denser, less porous product. By varyingthe pH of the collagen solution and the dPEG, solution, compositions maybe producing over a wide range of fibrillar content. If desired, thedenser formulations may be cast or molded into any shape desired, forexample into sheets or membranes, into tubes or cylinders, into cords orropes, and the like.

Breast Implants

Collagen-polymer conjugates can also be used as coatings for breastimplants. The surface of a standard silicone-shell implant can bechemically derivatized to provide active binding sites for di- ormultifunctional PEG-collagen (collagen-PEG-silicone). The presence ofthe collagen coating bound directly to the silicone via PEG will serveto reduce scar tissue formation and capsular contracture. Unlike typicalcoated breast implants, scar tissue will not be able to grow between thecollagen coating and the surface of the implant itself.

Alternatively, PEG-collagen can be formed into a hollow sphere for useas a breast implant shell. The shell can then be filled with aradiolucent material, such as triglycerides, to facilitate mammography.

Coated Medical Devices

The injectable formulations (gels or solutions) may be used to coatimplants, catheters, tubes (e.g., for blood vessel replacement), meshes(e.g., for tissue reinforcement), and the like. PEG-collagenformulations can also be used to coat platinum wires, which can then beadministered to the site of an aneurysm via catheter. Gels may beprepared by reducing the polymer concentration or reducing the reactiontime. CIS is the preferred starting material where the desiredproperties are high density, rigidity, viscosity, and translucence.However, one may substitute fibrillar collagen (preferably atelopeptidefibrillar collagen such as ZCI) and obtain products which are moreopaque, more flexible, and more susceptible to colonization by cellsafter implantation. CIS-based materials are presently preferred forcoating articles to be implanted, such as catheters and stress-bearingbone implants.

Conjugate and Cytokines

Compositions of the invention containing cytokines such as EGF and TGF-βare prepared by mixing an appropriate amount of the cytokine into thecomposition, or by incorporating the cytokine into the collagen prior totreatment with activated PEG. By employing an appropriate amount ofdifunctional PEG, a degree of crosslinking may be established, alongwith molecules consisting of collagen linked to a cytokine by asynthetic hydrophilic polymer. Preferably, the cytokine is first reactedwith a molar excess of dPEG* in a dilute solution over a 3 to 4 hourperiod. The cytokine is preferably provided at a concentration of about1 μg/mL to about 5 mg/mL, while the dPEG* is preferably added to a finalconcentration providing a 30 to 50-fold molar excess. The resultingconjugated cytokineis then added to an aqueous collagen mixture (about 1to about 60 mg/mL) at pH 7-8 and allowed to react further. The resultingcomposition is allowed to stand overnight at ambient temperature. Thepellet is collected by centrifugation, and is washed with PBS byvigorous vortexing in order to remove non-bound cytokine.

Membranous Forms

Flexible sheets or membranous forms of the collagen-polymer conjugatemay be prepared by methods known in the art, for example, U.S. Pat. Nos.4,600,533; 4,412,947; and 4,242,291. Briefly, high concentration (10-100mg/mL) CIS or fibrillar collagen (preferably atelopeptide fibrillarcollagen, such as ZCI) is cast into a flat sheet container. A solutionof mPEG* (having a molecular weight of approximately 5,000) is added tothe cast collagen solution, and allowed to react overnight at roomtemperature. The resulting collagen-polymer conjugate is removed fromthe reaction solution using a sterile spatula or the like, and washedwith PBS to remove excess unreacted mPEG*.

The resulting conjugate may then be compressed under constant pressureto form a uniform, flat sheet or mat, which is then dried to form amembranous implant of the invention. More flexible membranous forms areachieved by using lower collagen concentrations and high polymerconcentrations as starting materials.

Less flexible membranous forms are prepared by using a dPEG, solutionrather than mPEG*. CIS, at room temperature, is mixed with a buffersolution and incubated at 37° C. overnight. The resulting gel iscompressed under constant pressure, dried, and desalted by washing. Theresultant membrane is then crosslinked by treating with dPEG,, washed,and then dried at low temperature.

Alternatively, CIS or fibrillar collagen (10-100 mg/mL) is cast into aflat sheet container. A solution of dPEG* (22-50% w/v) is added to. thecast collagen. The mixture is allowed to react over several hours atroom temperature. Shorter reaction times result in more flexiblemembranes. The resulting collagen-polymer membrane may be optionallydehydrated under a vacuum oven, lyophilization, or air-drying.

Sponges

Collagen-polymer conjugates may also be prepared in the form of sponges,by lyophilizing an aqueous slurry of the composition after conjugation.

B.2 Use and Administration

Compositions of the invention have a variety of uses. Malleable, plasticcompositions may be prepared as injectable formulations, and aresuitable for dermal augmentation, for example for filling in dermalcreases, and providing support for skin surfaces. Such compositions arealso useful for augmenting sphincter tissue, (e.g., for restoration ofcontinence). In such cases, the formulation may be injected directlyinto the sphincter tissue to increase bulk and permit the occludingtissues to meet more easily and efficiently. These compositions may behomogeneous, or may be prepared as suspensions of small microgelcollagen-polymer conjugate particles or beads which are delivered in anon-aqueous carrier. The beads/particles rehydrate and swell in situ.This has the advantage over commercial preparations in that less volumeof product is required to achieve the desired correction.

Surprisingly, one may administer the reaction mixture by injectionbefore crosslinking has completed. In this embodiment, an aqueouscollagen mixture is combined with a low-concentration dPEG* solution,mixed, and the combination injected or applied before the viscosityincreases sufficiently to render injection difficult (usually about 20minutes). Mixing may be accomplished by passing the mixture between twosyringes equipped with Luer lock hubs, or through a single syringehaving dual compartments (e.g., double barrel). The compositioncrosslinks in situ, and may additionally crosslink to the endogenoustissue, anchoring the implant in place. In this method, one can usecollagen (preferably fibrillar collagen) at a concentration of about10-100 mg/mL, although abut 30-80 mg/mL is preferred, most preferablyabout 33 mg/mL. The dPEG* concentration is preferably set at about 0.1to about 3%, although concentrations as high as 30% may be used ifdesired. The mixture is injected directly into the site in need ofaugmentation, and causes essentially no detectable inflammation orforeign body reaction. One may additionally include particulatematerials in the collagen reaction mixture, for example hydrogel orcollagen-dPEG beads, or hydroxyapatite/tricalcium phosphate particles,to provide a bulkier or more rigid implant after crosslinking.

Compositions of the invention (particularly crosslinked collagencompositions) are also useful for coating articles for implantation orrelatively long term residence within the body. Such surface treatmentrenders the object non-immunogenic, and reduces the incidence of foreignbody reactions. Accordingly, one can apply compositions of the inventionto catheters, cannulas, bone prostheses, cartilage replacement, breastimplants, minipumps and other drug delivery devices, artificial organs,and the like. Application may be accomplished by dipping the object intothe reaction mixture while crosslinking is occurring, and allowing theadherent viscous coating to dry. One may pour or otherwise apply thereaction mixture if dipping is not convenient. Alternatively, one mayuse flexible sheets or membranous forms of collagen,polymer conjugate towrap the object with, sealing corners and edges with reaction mixture.

In another embodiment, the object may be dipped in a viscouscollagen-in-solution bath, or in a fibrillar collagen solution until theobject is completely coated. The collagen solution is fixed to theobject by dipping the collagen-coated object into a dPEG* (pH 7)solution bath, and then allowing the collagen-polymer coated object todry. Alternatively, viscous collagen-in-solution is mixed with a dPEG*(pH 3) solution and polymerized rapidly, as described above. The objectis dipped in the acidic collagen-polymer solution, and cured by dippingthe coated object into a neutralizing buffer containing about 20% byweight dPEG* (pH 7), to result in a collagen-polymer coated object.

Compositions of the invention may be prepared in a form that is denseand rigid enough to substitute for cartilage. These compositions areuseful for repairing and supporting tissue which require some degree ofstructure, for example in reconstruction of the nose, ear, knee.,larynx, tracheaI rings, and joint surfaces. One can also replace tendon,ligament and blood vessel tissue using appropriately formedcartilaginoid material. In these applications, the material is generallycast or molded into shape: in the case of tendons and ligaments, it maybe preferable to form filaments for weaving into cords or ropes. In thecase of artificial blood vessels it may be advantageous to incorporate areinforcing mesh (e.g., nylon or the like).

Compositions of the invention containing cytokines are particularlysuited for sustained administration of cytokines, as in the case ofwound healing promotion. Osteoinductive factors and cofactors (includingTGF-β) may advantageously be incorporated into compositions destined forbone replacement, augmentation, and/or defect repair. Compositionsprovided in the form of a membrane may be used to wrap or coattransplanted organs, to suppress rejection and induce improved tissuegrowth. Similarly, one may coat organs for transplantation using acrosslinking reaction mixture of factor-polymer conjugates and collagen.Alternatively, one may administer antiviral and antitumor factors suchas TNF, interferons, CSFs, TGF-β, and the like for their pharmaceuticalactivities. The amount of composition used will depend upon the severityof the condition being treated, the amount of factor incorporated in thecomposition, the rate of delivery desired, and the like. However, theseparameters may easily be determined by routine experimentation, forexample by preparing a model composition following the examples below,and assaying the release rate in a suitable animal model.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the conjugates and formulations and implants containing suchconjugates and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, molecularweight, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1

(Preparation of Collagen-PEG)

(A) Monomethyl-PEG 50.00 (50 g, 10 mmol, Aldrich Chemical Co.) isdissolved in 1,2-dichoroethane (250 mL) and heated at reflux withglutaric anhydride (5 g) and pyridine (4 mL) under nitrogen for 3 days.The solution is then filtered and the solvent evaporated, and theresidue dissolved in water (100 mL) and washed with diethyl ether (2×50mL). The resulting PEG-glutarate is extracted from the water withchloroform (2×50 mL), and the chloroform evaporated to yield about 43 gof PEG-glutarate. The PEG-glutarate is then dissolved indimethylformamide (DMF, 200 mL) at 37° C., and N-hydroxysuccinimide (10%molar xs) added. The solution is cooled to 0° C., and an equivalentamount of dicyclohexylcarbodiimide added in DMF solution (10 mL). Themixture is left at room temperature for 24 hours, and then filtered.Cold benzene (100 mL) is then added, and the PEG-succinimidyl glutarate(PEG-SG) precipitated by adding petroleum ether (200 mL) at 0° C. Theprecipitate is collected on a sintered glass filter. Dissolution inbenzene, followed by precipitation with petroleum ether is repeatedthree times to provide "activated" PEG (PEG-SG).

Vitrogen 100® collagen in solution (400 mL, 1.2 g collagen, 0,004 mmol)was mixed with 0.2M phosphate buffer (44 mL) to elevate the pH to 7.4.Next, a three-fold molar excess of PEG-SG (6.00 g, 1.2 mmol) wasdissolved in water for injection (40 mL) and sterile-filtered. ThePEG-SG solution was then added to the collagen solution, and the mixtureallowed to stand at 17-22° C for about 15 hours. The solution was thencentrifuged, and the resulting pellet (25 g) of reconstituted fibrilscollected and washed with phosphatebuffered saline (PBS, 3×400 mL) toremove residual PEG. The resulting material has a solid, coherentelasticity, and may be picked up on a spatula (the equivalentnon-conjugated collagen, Zyderm® collagen implant is more fluid). Theresulting material may be diluted with PBS to provide a dispersionhaving 20.5 mg/mL collagen-PEG.

(B) Similarly, proceeding as in part (A) above but substitutingpolypropylene glycol and POE-POP block polymers for polyethylene glycol,the corresponding collagen-PPG and collagen-POE-POP compositions areprepared.

(C) Difunctional PEG 3400 (34 g, 10 mmol, Aldrich Chemical Co.) isdissolved in 1,2-dichoroethane (250 mL) and heated at reflux withglutaric anhydride (10 g) and pyridine (4 mL) under nitrogen for 3 days.The solution is then filtered and the solvent evaporated, and theresidue dissolved in water (100 mL) and washed with diethyl ether (2×50mL). The resulting PEG-diglutarate is extracted from the water withchloroform (2×50 mL), and the chloroform evaporated to yieldPEG-diglutarate. The PEG-diglutarate is then dissolved in DMF (200 mL)at 37° C., and N-hydroxysuccinimide (10% molar xs) added. The solutionis cooled to 0° C., and an equivalent amount of dicyclohexylcarbodiimideadded in DMF solution (10 mL). The mixture is left at room temperaturefor 24 hours, and then filtered. Cold benzene (100 mL) is then added,and the PEG-di(succinimidyl glutarate) (dPEG-SG) precipitated by addingpetroleum ether (200 mL) at 0° C. The precipitate is collected on asintered glass filter. Dissolution in benzene, followed by precipitationwith petroleum ether is repeated three times to provide "activated" dPEG(dPEG*).

Vitrogen 100® collagen in solution (400 mL, 1.2 g collagen, 0.004 mmol)was mixed with 0.2M phosphate buffer (44 mL) to elevate the pH to 7.4.Next, a three-fold molar excess of dPEG* (6.00 g, 1.2 mmol) wasdissolved in water for injection (40 mL) and sterile-filtered. The dPEG*solution was then added to the collagen solution, agitated, and themixture allowed to stand at 17°-22° C. for about 15 hours. The solutionwas then centrifuged, and the resulting pellet of reconstituted fibrilscollected and washed with PBS (3×400 mL) to remove residual dPEG*. Thepellet was then placed in a syringe fitted with a Luer lock hubconnected to a second syringe, and was passed between the syringes untilhomogeneous. The resulting material is a microgel or a particulatesuspension of random size fibrils in solution (microgel conjugate). Thematerial is a smooth, pliable, rubbery mass, with a shiny appearance.

(D) Preparation of Cartilaginoid Conjugates:

Approximately 20% by weight of dPEG* (pH 7) was added to collagen insolution (33.8 mg/mL), and incubated at 21° C. for about 16 hours. Theresulting conjugate was washed with 100 mL PBS 3-5 times over 12 hours.The resulting cartilaginoid non-fibrillar collagen-polymer conjugate(NFC-F cartilage) was a translucent solid with coherent elasticity. Theproduct contained approximately 20-80% fibrillar collagen.

Another NFC cartilage composition was prepared by mixing dPEG* solution(0.6 g, pH 3) with collagen in solution (33.8 mg/mL, pH 2). The mixturewas passed between two syringes joined by a Luer lock connector to forma homogenous solution. A solution of dPEG* (20% w/v) in a neutralizingbuffer was then added to result in a substantially non-fibrillarcollagen (NFC) cartilage material. The resulting product containedapproximately 1-40% fibrillar collagen.

Alternatively, fibrillar collagen may be used instead of CIS to producea cartilaginoid fibrillar collagen-polymer conjugate (F cartilage)having an opaque appearance and high fibrillar content. Such F cartilageis more porous and permeable than non-fibrillar collagen-polymerconjugates.

Example 2

(Characterization)

(A) Collagen-mPEG prepared in Example 1A was characterized and comparedwith Zyderm® collagen implant (ZCI), and glutaraldehyde-crosslinkedfibrillar collagen (GAX).

Extrusion:

This assay measured the force required to extrude the test compositionthrough a 30 gauge needle. The results are shown in FIG. 1. As can beseen from the graph of force required (in Newtons) versus plungertravel, ZCI was extruded smoothly, requiring a force of about 20-30Newtons. GAX was not extruded smoothly, as shown by the "spiking"exhibited in the force trace. At the plateau, GAX required about 10-15Nfor extrusion. In contrast, collagen-mPEG demonstrated a very lowextrusion force (8-10N), with little or no spiking.

Intrusion:

Intrusion is a measure of the tendency of a composition to "finger" orchannel into a porous bed, rather than remaining in a compact mass. Lowintrusion is preferred in augmentation of soft tissue, so that theinjected implant does not diffuse through the dermis and remains inplace.

A 1 mL syringe fitted with a 30 gauge needle was half-filled withsilicon carbide particles (60 mesh), simulating human dermis. The upperhalf of the syringe was filled with 0.5 mL test composition (GAX, ZCI,or collagen-mPEG) at 35 mg/mL. The plunger was then fitted, anddepressed. On depression, ZCI appeared at the needle, demonstratingintrusion through the silicon carbide bed. Syringes filled with GAX orcollagen-mPEG of the invention did not pass collagen, instead releasingonly buffer, demonstrating no intrudability.

Helicity:

The portion of each composition exhibiting nonhelical character wasmeasured using sensitivity to digestion with trypsin. Samples weretreated with the protease trypsin, which is capable of attacking onlyfragmented portions of the collagen protein. The extent of hydrolysis ismeasured by fluorescamine assay for solubilized peptides, and theresults are expressed as percentage non-helical collagen. The percentageof non-helical collagen was measured 30 minutes after the beginning ofthe digestion period. The results indicated that ZCI was 3-10%sensitive, GAX was 1-2% sensitive, and collagen-mPEG was about 1%sensitive. Sensitivity to trypsin may also correlate to sensitivity toendogenous proteases following implantation.

Collagenase Sensitivity:

The sensitivity of each composition to collagenase was also measured.ZCI was 65.2% digested, compared to 2.2% for GAX, and 45.8% forcollagen-mPEG.

Phase Transition:

The behavior of each composition vs. temperature was examined using adifferential scanning calorimeter. On heating, ZCI exhibited multiplepeaks at about 45 and 53° C. GAX exhibited a peak at 67°-70° C.Collagen-mPEG exhibited a peak at 56°-61° C.

Lysine Content:

The number of free lysines per mole was determined for each compositionusing TNBS to quantify reactive epsilon amino groups. ZCI exhibitedabout 30 lysines per (single helix) molecule (K/m), whereas GAXexhibited 26-27 K/m, and collagen-mPEG 21-26 K/m.

(B) Characterization of Crosslinked Collagen-Polymer Conjugates:

A collagen-dPEG conjugate prepared as described in Example 1C wascharacterized using differential scanning calorimetry (DSC). This testis a measure of the transition temperature during fragmentation of thecollagen molecule at a microscopic level. A lowering of the transitiontemperature indicates an increase in fragmentation in a manner similarto that measured by trypsin sensitivity.

The collagen-dPEG conjugate showed a single denaturational transition at56° C. by DSC, which is similar to the typical melting point of theCollagen-PEG conjugate prepared in Example 1A. In comparison, ZCI has amelting temperature of 45°-53° C. with multiple denaturationaltransitions, and GAX has a melting temperature of 67°-70° C. with asingle denaturational transition.

The extrusion test described in Example 2A could not be used tocharacterize the collagen-dPEG conjugate because the material was notextrudable through a 30 gauge needle.

Using the intrusion test described in Example 2A, the passage ofcollagen-dPEG was completely blocked at the silicon carbide bed, whichindicates high crosslinking between the collagen molecules and little orno intrudability.

Example 3

(Immunogenicity)

(A) Non-crosslinked PEG-Collagen:

This experiment was conducted to demonstrate the relative immunogenicityof a collagen-mPEG preparation of the invention versus acommercially-available bovine collagen formulation prepared fromessentially the same source material, and having a similar consistency.As both collagen preparations were prepared using atelopeptide collagen(which is only weakly immunogenic), the preparations were formulatedwith either complete Freund's adjuvant (CFA) or incomplete Freund'sadjuvant (IFA), to enhance the immune response. This is a severe test,designed to magnify any possible immune reaction.

Collagen-mPEG was prepared as in Example 1A above. Male Hartley guineapigs (11) were anesthetized and bled by heart puncture forpre-immunization serologic evaluation. Five animals were treated withtwo 0.1 mL intramuscular injections of Zyderm® collagen implant (ZCI)emulsified in CFA (1:9) in the left and right thighs. Another fiveanimals were treated in the same fashion, using collagen-PEG (35 mg/mL)emulsified in CFA. One animal was treated with collagen-PEG in IFA. Atday 14 following immunization, all animals were again bled by heartpuncture, and serum obtained for antibody titer determination (usingELISA). Serology was again performed at day 30.

On day 30, following collection of serum samples, each animal waschallenged intradermally with both ZCI and collagen-PEG (0.1 mL of each,one on each flank). Delayed-type hypersensitivity (DTH) was quantifiedas a measure of cell-mediated immunity. DTH was evaluated at 24, 48, and72 hours post-challenge by measuring the diameter of any wheal usingmicrometer calipers, and noting the extent of erythema and induration.Animals were then euthanized with CO₂, and the injection sites excisedand fixed in neutral, buffered formalin for histological study.

Serological results indicated reduced immunogenicity of collagen-PEG vs.ZCI. At day 14, 80% of ZCI immunized animals exhibited "positive"antibody responses (titer ≧160 at day 14), whereas 0% of thecollagen-PEG immunized animals exhibited positive responses. At day 30,all ZCI-immunized animals exhibited high antibody titers, whereas noneof the collagen-PEG-immunized animals (C-PEG) exhibited high titers. Thedata are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Immunogenicity                                                                                   Antibody Titer                                             Animal     Treatment     day 14  day 30                                       ______________________________________                                        1          ZCI           320     >2560                                        2          ZCI           320     1280                                         3          ZCI           2560    >2560                                        4          ZCI           320     >2560                                        5          ZCI           80      2560                                         6          C-PEG         0       0                                            7          C-PEG         0       160                                          8          C-PEG         40      640                                          9          C-PEG         0       20                                           10         C-PEG         0       640                                          11         C-PEG (IFA)   0       160                                          ______________________________________                                    

Responses to the DTH challenge also demonstrated that the collagen-mPEGof the invention is less immunogenic. Guinea pigs immunized with ZCI andchallenged with ZCI exhibited a wheal measuring 1.128±058 cm indiameter. Animals immunized with collagen-mPEG and challenged withcollagen-mPEG exhibited wheals measuring 0.768±0.036 cm. Animalsimmunized with ZCI and challenged with collagen-mPEG, or immunized withcollagen-mPEG and challenged with ZCI, developed wheals smaller than theZCI-immunized ZCI-challenged wheals. Responses measured at 48 and 72hours were essentially the same or lower than the 24 hour response foreach site. Erythema was essentially the same for all animals.

Histological studies showed that both materials exhibited comparableintrusion, fingering into the dermis and subcutaneous space. Sites ofintradermal challenge with ZCI in ZCI-immunized animals exhibited themost extensive inflammatory response, including a cellular infiltrate oflymphohistiocytic elements with eosinophils and occasional giant cells.Two of the implant sites demonstrated an erosive inflammation of theoverlying epidermis and eschar formation. Sites of intradermal challengewith collagen-mPEG in ZCI-immunized animals exhibited only a moderateassociated inflammatory infiltrate, with a marked reduction in acutecells and lymphoid elements. Histiocytes and giant cells were moreprevalent, and in some samples lined and colonized the implants heavily.Animals immunized with collagen-mPEG exhibited only slight to moderatereaction, with ZCI challenge sites accompanied by a modestlymphohistiocytic perivascular infiltrate with a few eosinophils andgiant cells. Collagen-mPEG challenge sites were typically accompanied bya minimal scattering of lymphoid cells near the associated vasculature.

(B) Crosslinked dPEG-Collagen Conjugates:

Collagen-dPEG conjugates were prepared as in Example 1D. The sampleswere implanted in the dorsal subcutis and as cranial onlays in rats.After implantation for 30 days in the subcutis, NFC cartilage and NFC-Fcartilage materials had a homogeneous microfibrillar structure. Mildcolonization by connective tissue cells occurred at the periphery of theNFC-F cartilage samples, and mild capsule formation was present. Nocolonization had occurred with the NFC cartilage material and mildcapsule formation was present. F cartilage had a very fibrous structurewith mild but frequently deep colonization by connective tissue cellsand sparse numbers of adipocytes. Trace amounts of capsule were presentin limited areas of the F cartilage samples. NFC cartilage materialstended to retain their pre-implantation shape, with sharply definededges, while the NFC-F cartilage samples tended to flatten over time anddevelop rounded profiles.

When implanted as cranial onlays, the appearance of each of thematerials was similar to that in the subcutis except that the samplestended to become anchored to the skull via integration of the capsule orsurrounding loose connective tissue with the periosteum.

All of the samples appeared to be biocompatible, have differing degreesof colonization by host tissues, and varying mechanical characteristics.

Example 4

(In situ Crosslinking)

A dPEG solution was prepared as described Example 1C above. Thefollowing samples were then prepared:

(1) 5 mg dPEG in 80 μL water, mixed with 0.5 mL fibrillar collagen (35mg/mL), to a final dPEG concentration of 1% by volume;

(2) 15 mg dPEG in 80 μL water, mixed with 0.5 mL fibrillar collagen (35mg/mL), to a final dPEG concentration of 3% by volume;

(3) Vitrogen® 100 collagen in solution;

(4) 5 mg dPEG in 80 μL water, mixed with 0.5 mL non-fibrillar collagen(35 mg/mL), to a final dPEG concentration of 1% by volume;

(5) 15 mg dPEG in 80 μL water, mixed with 0.5 mL non-fibrillar collagen(35 mg/mL), to a final dPEG concentration of 3% by volume;

(6) 5 mg dPEG in 0.5 ml PBS, to a final dPEG concentration of 1% byvolume; and

(7) GAX.

The dPEG solutions of Samples 1, 2, 4, and 5 were placed in a 1 mLsyringe equipped with a Luer lock fitting and connector, and joined toanother syringe containing the collagen material. The solutions weremixed by passing the liquids back and forth between the syringes severaltimes to form the homogeneous reaction mixture.

The syringe connector was then removed and replaced with a 27 gaugeneedle, and approximately 50 μL of the reaction mixture was injectedintradermally into each of 20 guinea pigs. Samples 3, 6, and 7 weresimilarly administered through a 27 gauge needle. At intervals up to 30days following injection, the treatment sites were harvested and studiedhistologically.

By 30 days, all of the materials appeared to be biocompatible. Samples 1and 2 displayed wide dispersion with an intermediate degree ofinterdigitation with dermal collagen fibers. Colonization by connectivetissue cells was moderate, and a trace of round cell infiltrate witheosinophils was seen.

Samples 3, 4 and 5 were highly dispersed and finely interdigitated withdermal collagen fibers. Colonization was mild to moderate, and tracelevels of round cell infiltration were seen.

Sample 6 had no detectable effects. Sample 7 occurred as large islandswith moderate colonization and trace to mild levels of inflammation.

Example 5

(Coating of Implants)

A collagen-dPEG reaction mixture was prepared as described in Example 1Cabove. A titanium implant was dipped into the reaction mixtureapproximately 20 minutes after crosslinking was initiated. The implantwas then allowed to finish crosslinking, and dry overnight.

Example 6

(Collagen-Polymer-Growth Factor Conjugates)

(A) A conjugate containing crosslinked collagen-dPEG-TGF-β2 was preparedas follows:

A solution of TGF-β2 and ¹²⁵ I-TGF-β2 (10⁵ cpm; 25 μL of 1 mg/mL) wasadded to a solution of dPEG* (4 mg) in CH₂ C₁₂ (100 μL), and the mixtureallowed to react for 2 (sample #3) or 35 (sample #5) minutes at 17° C.To this was added 2.5 mL of collagen solution (3 mg/mL atelopeptidenonfibrillar collagen), and the resulting mixture allowed to incubateovernight at ambient temperature. The pellet which formed was collectedby centrifugation to provide collagen-dPEG-TGF-β2.

(B) A composition based on fibrillar atelopeptide collagen was preparedas in part A above, but limiting TGF-β2/dPEG* reaction time to 2minutes, and substituting 7 mg of fibrillar collagen (precipitated fromcollagen in solution within 2 minutes prior to use) for collagen insolution.

(C) A composition containing dPEG-crosslinked collagen and free TGF-β2was-prepared as follows:

A solution of dPEG* (4 mg) in CH₂ Cl₂ (100 μL), was added to 2.5 mLof-CIS (3 mg/mL atelopeptide nonfibrillar collagen), and the resultingmixture allowed to incubate overnight at ambient temperature. The pelletwhich formed was washed to remove unreacted dPEG*, and 25 μg of TGF-β2mixed in to provide collagen-dPEG+TGF-β2.

(D) The degree of TGF-β2 binding was determined as follows:

Each composition prepared in parts A-C above was washed six times with0.5 mL of buffer (0.02 M phosphate buffer, 0.1% BSA) by vigorousvortexing followed by centrifugation in order to remove non-boundTGF-β2. The pellet and supernatants were collected at each time ofwashing, and were counted. FIG. 2 demonstrates the release rate of thecompositions of part A (open circles) and part B (filled circles) versusthe simple mixture prepared in part C (x's), showing the number ofcounts release a as a function wash cycle. As shown in the figure, theTGF-β2 in the simple mixture is quantitatively released within about 6washings, while approximately 40% of the TGF-β2 is retained in thecompositions of part B and 50% is retained in the compositions of partA.

(E) The biological activity of the materials prepared above was assayedas follows:

Compositions prepared according to part A (CIS-dPEG-TGF-β2)(TGF-β2/dPEG* reaction time of 12 minutes) and part C (CIS-dPEG+TGF-β2)were prepared, as well as a control prepared according to part C withoutTGF-β2 (CIS-dPEG). The samples were washed in PBS/BSA eight times asdescribed in part D, then washed an additional three times in fetalbovine serum (Gibco) at 37° C. This washing protocol resulted invisually detectable material loss, so remaining TGF-β2 content wasdetermined-by counting the remaining ¹²⁵ I. TGF-β2 activity was thenassayed by ELISA. The results are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Retention of Biological Activity                                                             .sup.125 I                                                                             remaining  O.D.                                       Sample         Counts   TGF-β2(μg)                                                                       (414 nm)                                   ______________________________________                                        CIS-dPEG         0      0          0.015                                                                         0.015                                      CIS-dPEG + TGF-β2                                                                        2775    0.5-1.0    0.029                                                                         0.035                                      CIS-dPEG-TGF-β2                                                                         42604    7.4        0.102                                                                         0.082                                      ______________________________________                                    

The data demonstrates that the TGF-β1 retained in the compositions ofthe invention remains in a substantially active form.

Example 7

(Formulations)

(A) A formulation suitable for implantation by injection was prepared bysuspending collagen-PEG in sterile water for injection, at 35 mg/mL. Thecharacteristics of the resulting formulation are described in Example 2above.

(B) A formulation useful for repair of stress-bearing bone defects(e.g., fractures, nonunions, and the like) may be prepared by mixingcollagen-PEG of the invention with a suitable particulate, insolublecomponent. The insoluble component may be fibrillar crosslinkedcollagen, gelatin beads, polytetrafluoroethylene beads, silicone rubberbeads, hydrogel beads, silicon carbide beads, mineral beads, or glassbeads, and is preferably a calcium mineral, for example hydroxyapatiteand/or tricalcium phosphate.

Solid formulations were prepared by mixing Zyderm® II (65 mg/mLcollagen) or collagen-mPEG (63 mg/mL) with particulate hydroxyapatiteand tricalcium phosphate (HA+TCP) and air drying to form a solid blockcontaining 65% HA by weight. Optionally, blocks were heat-treated byheating at 75° C. for 10 hours. The resulting blocks were hydrated in0.13M saline for 12 hours prior to testing.

On standing, it was observed that Zyderm®-HA+TAP (Z-HA) compositionsseparated into three phases, whereas PEG-collagen-HA+TCP (PC-HA)compositions remained single phase.

Each block was elongated by 5%, and its stress relaxation monitored for1 minute after release. After this test, each block was subjected toconstant elongation at a constant 1 cm/min until failure. The resultsare shown in Table 3:

                  TABLE 3                                                         ______________________________________                                        Mechanical Strength                                                           Stress Relaxation    Constant Extension                                              Peak    Constant t.sub.1/2                                                                            Rupture                                                                              Extension                               Sample Force   Force    (min)  Force  at Rupture                              ______________________________________                                        Z-HA   1.5     1.1      0.04   2.6    11.0%                                   (air)  --      --       --     2.6    15.3%                                   Z-HA   1.5     1.1      0.06   --     --                                      (heat) 1.4     1.0      0.07   3.4    14.0%                                   PC-HA  2.6     1.8      0.06   5.5    12.3%                                   (air)  2.8     2.1      0.08   5.4    11.7%                                   PC-HA  3.3     2.6      0.04   5.4    12.0%                                   (heat) 3.6     2.7      0.06   5.4    20.3%                                   ______________________________________                                         All forces reported in newtons. Extension at rupture (strain) reported in     percent extension.                                                       

The data demonstrate that collagen-polymer forms HA+TCP compositionsexhibiting substantially greater tensile strength. Thus, one can prepareimplant compositions with collagen-polymer which are substantiallystronger than compositions employing the same amount of non-conjugatedcollagen, or may reduce the amount of collagen-polymer employed to forma composition of equal strength.

The instant invention is shown and described herein at what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

What is claimed:
 1. An ophthalmic device comprising a conjugatecomprising nonfibrillar collagen chemically conjugated by an etherlinkage to a synthetic hydrophilic polymer.
 2. The device of claim 1,wherein the device is a lenticule.
 3. The device of claim 1, wherein thedevice is a corneal shield.
 4. The device of claim 1, wherein saidsynthetic hydrophilic polymer is bound to an available lysine residue onsaid collagen.
 5. The device of claim 4, wherein said synthetichydrophilic polymer molecules are bound to 10-50% of said availablelysine residues.
 6. The device of claim 5, wherein said synthetichydrophilic polymer molecules are bound to 20-30% of said availablelysine residues.
 7. The device of claim 4, wherein the ratio of collagenmolecules to polymer molecules is about 1:1 to about 1:20.
 8. The deviceof claim 1, wherein said polymer is a functionally activatedpolyethylene glycol.
 9. The device of claim 8, wherein said polymer hasa weight average molecular weight of about 100 to about 20,000.
 10. Thedevice of claim 8, wherein said conjugate has the general structuralformula collagen-NH--C(═O)--(CH₂)_(n) --O--PEG--O--(CH₂)_(n)--C(═O)--NH-collagen, wherein n is an integer from 0 to 4.